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Updated: Jun 2, 2026
A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
Published on: June 6, 2018
A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum
Hakeenah F Brown1, Ling Wang, Sudip Khadka
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Abstract:
Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.
Insights
We developed new methods to improve the yeast two-hybrid assay for studying Plasmodium falciparum protein interactions. These techniques enable better gene expression and interaction validation, aiding malaria research.
Area of Science:
- Molecular Biology
- Parasitology
Background:
- Studying Plasmodium falciparum protein-protein interactions is crucial for understanding malaria.
- Traditional yeast two-hybrid assays are limited by poor P. falciparum gene expression in yeast.
Purpose of the Study:
- To develop improved methods for screening Plasmodium falciparum protein-protein interactions using the yeast two-hybrid assay.
- To validate identified interactions using orthogonal experimental approaches.
Main Methods:
- Generated gene fragments using random DNAse I digestion and PCR-based methods for yeast two-hybrid screening.
- Expressed proteins in wheat germ extracts for validation.
- Confirmed interactions via split-luciferase assay and co-purification experiments.
Main Results:
- Successfully expressed most P. falciparum gene portions in yeast.
- Identified a putative interaction between PfMyb2 and PFC0365w.
- Validated the interaction using multiple independent assays.
Conclusions:
- The developed methods enhance the utility of yeast two-hybrid assays for P. falciparum research.
- Improved screening and validation techniques facilitate the study of parasite protein interactions.
